More Than One Amine Oxidase is Involved in the Metabolism by Yeasts of Primary Amines Supplied as Nitrogen Source

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Journal of General icrobiology (98), 8, 99-996.

1 Journal of General icrobiology (98), 8, Printed in Great ritain 99 ore Than One mine Oxidase is Involved in the etabolism by Yeasts of Primary mines Supplied as itrogen Source y JEFFREY GREE, GEOFFREY W. HYWOOD D PETER J. LRGE* Department of iochemistry, University of Hull, Hull HU6 7RX, U.K. (Received September 98 ) Twenty-seven yeast species were tested for growth on methylamine and n-butylamine as sole nitrogen sources. Five species (including Saccharomyces cerevisiae) failed to grow on either amine, and a further five grew only on n-butylamine. The remainder grew on both amines. mine oxidase activity was detected in extracts of all the strains which could grow on amines, but was generally absent from cells grown on ammonia or nitrate. From measurements in cell-free extracts of oxidase activity for a number of different amines, it is concluded that most such strains have at least two amine oxidases of different substrate specificity, and that the substrate specificity also varies between different yeast species. ased on these observations, four different groups of yeasts could be distinguished. Formaldehyde dehydrogenase activity was elevated in cells grown on amines or nitrate compared with those grown on ammonia, and activity was generally higher in cells grown on methylamine than in those grown on n-butylamine or nitrate. ITRODUCTIO That yeasts can grow on amines as sole nitrogen source has been known for many years, and ethylamine utilization has been used as a diagnostic tool since it was proposed by Van der Walt (96). The extent of the capabilities of yeasts to utilize amines as sole nitrogen source, however, was not fully realized until Van Dijken & os (98 ) showed that 86 % of the 46 strains of yeast from various genera tested were able to use at least one amine as sole nitrogen source, and 3% could use all of the eight amines tested. It is generally assumed that utilization of amines for growth involves an amine oxidase, and this has been shown to be true for growth of Trichosporon sp. on trimethylamine (Yamada et al., 966), growth of Candida utilis and Hansenula polymorpha on methylamine (Zwart et al., 98) and growth of Candida boidinii on a number of different amines (Haywood & Large, 98). We have examined 7 yeast strains in an attempt to answer the following questions. () re amine oxidases always present when yeasts are grown on primary aliphatic amines as sole nitrogen source? () In yeasts grown on a single amine substrate, what other amines can be oxidized by cell-free extracts? (3) How many different amine oxidases are present during growth on any single amine substrate? (4) Does the nature or number of amine oxidases present in a cell change when the growth substrate is changed? In addition, evidence (Van Dijken et al., 979; Zwart et al., 98; Van Dijken & os, 98; Haywood & Large, 98 ) suggests that formaldehyde dehydrogenase (EC...) activity is elevated in cells grown on methylated amines compared with cells grown on ammonium salts. We investigate here whether this is a general phenomenon in yeasts and also whether it occurs when other non-ammonium nitrogen sources are used. -87/8/ SG IP: On: Fri, 3 ov 8 7::3

99 J. GREE, G. W. HYWOOD D P. J. LRGE ETHODS Organisms used. With two exceptions, the yeast strains were gifts from Dr J. P. van Dijken (Delft, The etherlands); 8 of the 7 were type strains from the Centraalbureau voor Schimmelcultures (Yeast Division) in Delft (CS).

2 99 J. GREE, G. W. HYWOOD D P. J. LRGE ETHODS Organisms used. With two exceptions, the yeast strains were gifts from Dr J. P. van Dijken (Delft, The etherlands); 8 of the 7 were type strains from the Centraalbureau voor Schimmelcultures (Yeast Division) in Delft (CS). The nomenclature of the strains used is that of arnett et al. (979) except that former Torulopsis species have been transferred to the genus Candida as proposed by Yarrow & eyer (978). The designatory abbreviations for other culture collections are: CYC, ational Collection of Yeast Cultures (orwich, U.K.); G, culture collection of icrobiology Laboratory, University of Groningen, The etherlands. aintenance. Strains were maintained on slopes containing % (w/v) malt extract and % (w/v) agar. Growth of cells. Yeasts were grown on the defined medium previously described (Haywood & Large, 9Sl), with glucose ( m) as carbon source and one of the following as nitrogen source: m-methylamine.hc, m-(h,),so,, m-ko, or m-n-butylamine.hc. In most cases, the cells were in the exponential growth phase (s63 between and ) when they were harvested. They were washed with m-potassium phosphate ph 7. and stored at -8 C until used. Preparation of extracts and enzyme assays. Extracts were prepared in the French press as described by Haywood & Large (98 I), and enzymes were assayed by the following methods. ethylamine, rz-butylamine, isobutylamine, benzylamine and putrescine oxidases were assayedby the peroxidase-coupled spectrophotometric assay of Haywood & Large (98), with the substrates at the following final concentrations: methylamine, 3.3 m; n-butylamine, m; isobutylamine, m; benzylamine,.33 m; putrescine, m. Formaldehyde dehydrogenase (EC...) and formate dehydrogenase (EC...) were assayed by the method of Van Dijken et al. (976), and isocitrate dehydrogenase (DP-linked, EC...4) by the method of ergmeyer (974). The formaldehyde and formate dehydrogenase activities were corrected by addition of the DH oxidase activity measured at the same ph. In some cases (marked 3: in Table ) this DH oxidase activity was so high that no dehydrogenase activity could be measured. Poiyacrylarnide gel electrophoresis. Electrophoresis of crude extracts and staining for amine oxidase activity were performed as described by Haywood & Large (98 ). This method can detect extremely low levels of amine oxidase activity. Protein. This was determined by the method of radford (976). RESULTS D DISCUSSIO The yeast strains tested were selected on the basis of the results of Van Dijken & os (98). They fell into three categories: (a) those which grew on both methylamine and n-butylamine, (b) those which grew on n-butylamine but not on methylamine and (c) those which failed to grow on either amine as sole nitrogen source. In all but three cases our observations as to which amines could be used for growth agreed with those of Van Dijken & os (98 ). In category (c) were Cryptococcus albidus CS 4, Pichia tannicola CS 66, Saccharomyces cerevisiae CYC 4*, Schizosaccharomyces japonicus var. japonicus CS 34 and Candidu (ToruZopsis) glabrata CS 663*. (n asterisk, both here and in Table denotes a strain which is not a type strain.) This category (c) was not examined further. The various enzyme activities examined were the oxidases for five primary amines (methylamine, n-butylamine, isobutylamine, benzylamine and putrescine), formaldehyde dehydrogenase, formate dehydrogenase and DP-linked isocitrate dehydrogenase. The latter was selected because it would not be expected to show significant variation when the nitrogen source was changed. Expression of amine oxidase activity by the yeast strains During growth on nitrate or ammonium as nitrogen source, no amine oxidase activity could be detected, except in two cases, Hansenula polymorpha and Candida nemodendra, where low levels were detected in cells grown on ammonia (Table ). This does not affect our conclusion that ammonia and nitrate both repress amine oxidase activity. During growth on amines, however, in all cases oxidase activity for at least one of the five amines tested was found. In a few cases no activity could be detected with the amine which had been used as nitrogen source (see below). From work on Candida boidinii (Haywood & Large, 98) it is known that yeasts may possess more than one amine oxidase capable of oxidizing n-butylamine. In Candidu boidinii one of these enzymes ( benzylamine oxidase ) is much less effective in oxidizing methylamine, IP: On: Fri, 3 ov 8 7::3

3 ultiple amine oxidases in yeasts 993 while the second ( methylamine oxidase ) is much less effective in oxidizing benzylamine. The strategy used was to grow the yeasts on n-butylamine and methylamine and to measure the levels of oxidase activity for methylamine, benzylamine and n-butylamine. The increase of n-butylamine oxidase activity in cells grown on n-butylamine and the increase of methylamine oxidase in cells grown on methylamine in most of the strains tested (Table ) suggests that enzyme multiplicity occurs in almost all cases. The only paradoxical result occurs in the genus Pichia where both activities are lower in cells grown on methylamine. The existence of multiple amine oxidase enzymes was also borne out by the presence of multiple bands in activity stains on polyacrylamide gels (Table l), although these data have to be treated with caution because pure yeast amine oxidases are known to give multiple bands (Haywood & Large, 98 ). The activity stains, however, did allow detection of amine oxidase activity in those extracts where no activity could be measured in the assays with substrates on which the cells had been grown. There are five instances of this in Table. Such failure to measure activity may have been due to having harvested the cells at the wrong stage in the growth cycle, or to use of sub-optimal assay conditions, because all such experiments were repeated at least once, giving the same result. The assay conditions used have been established as optimal for Candida boidinii, but may of course be different in other species. From the amine oxidase data in Table, we may divide the organisms into four classes: (i) Species with high methylamine, benzylamine and n-butylamine oxidase activities during growth on one or both amine substrates: Candida lipolytica, C. nagoyaensis, C. utilis, Hansenula minuta, H. polymorpha, Pichia pastoris, P. pinus, Sporopachydemia cereana and Trigonopsis variabilis. (ii) Species with high methylamine and n-butylamine oxidase activities but low benzylamine oxidase activity: Candida boidinii and C. nemodendra. (iii) Species with high methylamine oxidase activity but low n-butylamine and benzylamine oxidase activities : R hodotorula graminis and Sporobolomyces albo-rubescens. (iv) Species with low methylamine oxidase activity but high n-butylamine and benzylamine oxidase activities : Candida steaiolytica, Kluyveromyces fragilis, K. lactis, K. phaseolosporus, and all the organisms in category (b) in Table. Further investigation will be necessary to establish whether these groupings really reflect genuine differences, but what is clearly shown is that not only are there multiple enzymes, but that these enzymes differ in substrate specificity. This was confirmed by the finding of putrescine oxidase activity in only five organisms: Candida nagoyaensis (grown on methylamine), C. steaiolytica (grown on both amines), C. utilis (grown on butylamine), Pichia pastoris (grown on butylamine) and Sporopachydemia cereana (grown on methylamine). In methylamine-grown Candida steatolytica, putrescine oxidase was the only oxidase activity which could be detected by activity measurement. In Carzdida boidinii it is known (Haywood & Large, 98 ) that neither methylamine oxidase nor benzylamine oxidase can oxidize putrescine. Further evidence of differences in substrate specificity is given by activity with isobutylamine (only one of the two amine oxidases in Candida boidinii will oxidize this substrate). ost of the organisms in group (i) (except in the two Hansenula species) and all those in category (6) of Table had detectable isobutylamine oxidase activity (although high levels were only found in Candida nagoyaensis, C. boidinii, Sporopachydemia cereana and Trigonopsis variabilis). It may be significant that all species without isobutylamine oxidase activity were ascospore-forming strains. The occurrence of more than one amine oxidase, already established for Candida boidinii (Haywood & Large, 98) and for the fungus Phycomyces blakesleeanus (Hofmann & Hilgenberg, 979), thus proves to be a general phenomenon in yeasts. lthough the organisms in category (b) had very low methylamine oxidase activity, this is not the sole cause of their failure to grow on methylamine, because this was also true of Candida steatolytica and the three Kluyveromyces spp. in category (a) which grew slowly on methylamine. It may perhaps be coupled with inability to transport methylamine. IP: On: Fri, 3 ov 8 7::3

4 Table. Enzyme activities present in yeasts grown on primary arnines as sole nitrogen sources Cells were grown on n-butylamine (group b) or n-butylamine and methylamine (group a) and disrupted as described in ethods. In a number of cases cells were also grown on ammonium sulphate or potassium nitrate. Crude extracts were prepared with a French press and assayed spectrophotometrically for enzyme activities and protein concentration. Some extracts were also examined for amine oxidase activity on polyacrylamide gels after electrophoretic separation (-, no data available). bbreviations used to denote the various nitrogen sources for growth:, ammonium sulphate;, n-butylamine;, methylamine;, potassium nitrate. Organism and strain o. of amine Specific activity [nmol min- (mg protein)- ) itrogen oxidase I \ source for bands ethylamine enzylamine n-utylamine Formaldehyde Isocitrate growth on gel oxidase oxidase oxidase dehydrogenase dehydrogenase Candida boidinii CS 777* C. lipolytica CS 74* C. nagoyaensis CS 683 C. nemodendra CS 68 C. steatolytica CS 839 C. utilis CYC 3* Hansenula minuta CS 78 H. polymorpha CS 473 (a) Species capable of growth on both n-butylamine and methylamine as nitrogen source t t t t t I * I 43 * \o W P e U P : tl cd 4 r? El Q IP: On: Fri, 3 ov 8 7::3

5 Kluyveromyces fragilis G7 4* K. lactis CS 39; K. phaseolosporus CS 3 Pichia pastoris CS 74 P. pinus CS 98* R hodotorula graminis CYC 98; Sporobolomyces albo-rubescens CS 48 Sporopachydemia cereana CS 6644 Trigonopsis variabilis CS 4 Candida insectalens CS 636 Kluyveromyces wickerhamii CS 7 Pichia media CS P.polymorpha CS 86 Trichosporon melibiosaceum CS 687 t i (b) Organisms capable of growth on n-butylamine but not methylamine as nitrogen source * ot a type strain. t Putrescine oxidase activity was also found (see text). 3: ctivity lower than DH oxidase and therefore undetectable IP: On: Fri, 3 ov 8 7::3

6 996 J. GREE, G. W. HYWOOD D P. J. LRGE The data do not allow us to determine precisely how many amine oxidase isoenzymes exist in the different strains. This will require either enzyme purification or more sophisticated identification methods for crude extracts. Partial purification of putrescine oxidase activity from Pichia pastoris and Candida utilis has shown that in both these organisms the same enzyme can oxidize putrescine, n-butylamine and benzylamine (unpublished results). Formaldehyde and formate dehydrogenase activity The results in Table support the suggestion of Van Dijken & os (98) that enzymes oxidizing formaldehyde are widespread in yeasts and are elevated in activity in cells grown on methylated amines. ctivities of formaldehyde dehydrogenase were generally significantly higher in cells grown on methylamine than in n-butylamine-grown cells, which themselves had activities higher than in cells grown on ammonia. The activities of formate dehydrogenase were so low as to be in most cases below the level of DH oxidase activity and so undetectable (not shown in Table ). This is to be expected since formate dehydrogenase activity only rises during the stationary phase of cell growth on methylamine (Zwart et al., 98). That the derepression of formaldehyde dehydrogenase observed in most cases in cells grown on n-butylamine compared with the levels of the enzyme in ammonia-grown cells is not directly concerned with amine metabolism is shown by the fact that in most cases the enzyme also seemed to be derepressed in cells grown on nitrate. Derepression of formaldehyde dehydrogenase during growth on glucose plus ammonia at low dilution rates was shown for Hansenulu polymorpha and Kloeckera sp. by Egli et al. (98). The relatively slight changes in DP-linked isocitrate dehydrogenase with nitrogen source in most cases suggests that the changes observed in the other enzymes examined are indeed of physiological significance. We thank Dr J. P. van Dijken for valuable discussions and suggestions. This work was supported by grant GR//3687 from the Science and Engineering Research Council, which is gratefully acknowledged. REFERECES REIT, J.., PYE, R. W. & YRROW, D. (979). Guide to Identifying and Classifying Yeasts. Cambridge: Cambridge University Press. ERGEYER, H. U. (editor) (974). ethods of Enzymatic nalysis, nd English edn, pp Weinheim: Verlag-Chemie. RDFORD,.. (976). rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. nalytical iochemistry 7, EGLI, TH., V DIJKE, J. P., VEEHUIS,., HRDER, W. & FIECHTER,. (98). ethanol metabolism in yeasts: regulation of the synthesis of catabolic enzymes. rchives of icrobiology 4, -. HYWOOD, G. W. & LRGE, P. J. (98). icrobial oxidation of amines. Distribution, purification and properties of two primary-amine oxidases from the yeast Candida boidinii grown on amines as sole nitrogen source. iochemical Journal 99, 87-. HOF, F. & HILGEERG, W. (979). minooxidasen in Phycomyces blakesleeanus und ihre edeutung fur die Indol-3-essigsaurebiosynthese des Pilzes. ericht der Deutschen botanischen Gesellschaft 9, 6-6. V DLIKE, J. P. & os, P. (98). Utilization of amines by yeasts. rchives of icrobiology 8, V DIJKE, J. P., OTTO, R. & HRDER, W. (976). Growth of Hansenula polymorpha in a methanollimited chemostat. Physiological responses due to the involvement of methanol oxidase as a key enzyme in methanol metabolism rchives of icrobiology, V DLIKE, J. P., VEEHUIS,., ZWRT, K., LRGE, P. J. & HRDER, W. (979). Utilisation of methylamine by yeasts. Society for General icrobiology Quarterly 6, 7. V DER WLT, J. P. (96). Utilization of ethylamine by yeasts. ntonie van Leeuwenhoek 8,9-96. YD, H., KUGI, H., UWJI, T. & OGT, K. (966). Trimethylamine metabolism. I. onomethylamine oxidase. emoirs of the Research Institute for Food Science, Kyoto University 7, -4. YRROW, D. & EYER, S.. (978). proposal for amendment of the diagnosis of the genus Candida erkhout nom.cons. International Journal of Systematic acteriology 8, 6-6. ZWRT, K., VEEHUIS,., V DIJKE, J. P. & HRDER, W. (98). Development of amino oxidase-containing peroxisomes in yeasts during growth on glucose in the presence of methylamine as the sole source of nitrogen. rchives of icrobiology 6, 7-6. IP: On: Fri, 3 ov 8 7::3

AN ELECTROPHORETIC COMPARISON OF ENZYMES IN STRAINS OF SPECIES IN THE FISSION YEAST GENERA SCHJZOSACCHAROMYCES, 0CTOSPOROMYCES, AND HA SEGA WAEA1

J. Gen. Appl. Microbiol., 33, 363-369 (1987) AN ELECTROPHORETIC COMPARISON OF ENZYMES IN STRAINS OF SPECIES IN THE FISSION YEAST GENERA SCHJZOSACCHAROMYCES, 0CTOSPOROMYCES, AND HA SEGA WAEA1 YUZO YAMADA,